U.S. patent application number 13/496437 was filed with the patent office on 2012-09-27 for partial peptide of lacritin.
This patent application is currently assigned to SENJU PHARMACEUTICAL CO., LTD.. Invention is credited to Mitsuyoshi Azuma, Takeshi Nakajima, Tomoko Nakajima.
Application Number | 20120245102 13/496437 |
Document ID | / |
Family ID | 43758803 |
Filed Date | 2012-09-27 |
United States Patent
Application |
20120245102 |
Kind Code |
A1 |
Nakajima; Takeshi ; et
al. |
September 27, 2012 |
PARTIAL PEPTIDE OF LACRITIN
Abstract
The invention provides a polypeptide containing an amino acid
sequence, which is a particular partial sequence of lacritin and is
characterized by gyro-modification of N-terminal glutamine. The
polypeptide promotes adhesion between a cell and extracellular
matrix and is stable in aqueous solution.
Inventors: |
Nakajima; Takeshi; ( Hyogo,
JP) ; Nakajima; Tomoko; ( Hyogo, JP) ; Azuma;
Mitsuyoshi; ( Hyogo, JP) |
Assignee: |
SENJU PHARMACEUTICAL CO.,
LTD.
Osaka-shi, Osaka
JP
|
Family ID: |
43758803 |
Appl. No.: |
13/496437 |
Filed: |
September 16, 2010 |
PCT Filed: |
September 16, 2010 |
PCT NO: |
PCT/JP2010/066611 |
371 Date: |
April 10, 2012 |
Current U.S.
Class: |
514/20.8 ;
435/375; 530/324 |
Current CPC
Class: |
C07K 14/475 20130101;
A61K 9/08 20130101; A61P 43/00 20180101; A61K 9/0048 20130101; A61P
27/02 20180101; A61K 38/00 20130101 |
Class at
Publication: |
514/20.8 ;
530/324; 435/375 |
International
Class: |
A61K 38/17 20060101
A61K038/17; C12N 5/071 20100101 C12N005/071; A61P 27/02 20060101
A61P027/02; C07K 14/47 20060101 C07K014/47 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 16, 2009 |
JP |
2009-215030 |
Claims
1. A polypeptide comprising the amino acid sequence of SEQ ID NO:
1.
2. An agent for promoting cell adhesion, comprising the polypeptide
of claim 1.
3. A method of promoting cell adhesion, comprising contacting an
effective concentration of the polypeptide of claim 1 with a
cell.
4. A medicament comprising the polypeptide of claim 1 as an
effective ingredient.
5. The medicament of claim 4, which is aqueous liquid.
6. The medicament of claim 4, which is used for the prophylaxis or
treatment of corneal epithelium disorder.
7. A method for the prophylaxis or treatment of corneal epithelium
disorder in a patient in need thereof, which comprises
administering an effective amount of the polypeptide of claim
1.
8. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1
for promoting cell adhesion.
9. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1
for the prophylaxis or treatment of corneal epithelium disorder.
Description
TECHNICAL FIELD
[0001] The present invention relates to a polypeptide having a
particular partial sequence of lacritin which is a protein in the
tear fluid. Specifically, the present invention relates to a
modified partial peptide of lacritin.
BACKGROUND ART
[0002] It is known that cell-extracellular matrix adhesion is
involved in various functions such as cell survival, motility and
the like. This is a process essential for controlling the normal
development of individual, maintenance of tissues, or recovery from
damage or infection. An abnormality in the signaling pathway based
on such cell adhesion sometimes leads to abnormal development,
circulatory diseases or transformation or metastasis of the
cells.
[0003] In addition, it has been reported that when the
cell-extracellular matrix adhesion is inhibited, the cells reach
cell death called "anoikis", and therefore, adhesion to an
extracellular matrix is important for the survival of the cells
(see non-patent document 1).
[0004] Lacritin is a protein identified as a tear secretion
promoting factor or a growth-factor-like protein (see patent
documents 1 and 2 and non-patent document 2). For lacritin, the
following 1) to 5) are reported:
1) Lacritin has an activity as a growth factor for a corneal
epithelial cell and a lacrimal gland acinar cell. 2) Lacritin shows
a promoting effect on tear protein secretion. 3) Lacritin is
expressed in a cell derived from tissues such as the lacrimal
gland, parotid gland, minor salivary gland, submandibular gland,
thyroid gland, mammary gland and corneal epithelium. 4) Eye drops
containing lacritin are likely to be useful in the treatment Of
ocular diseases such as dry eye syndrome, Sjogren's syndrome, and
corneal epithelial wounds. 5) Compounds that bind to lacritin or
lacritin receptors can be screened for using a cell expressing a
lacritin receptor with a lacritin-dependent calcium signal as an
index.
[0005] In addition, it has been reported that lacritin or a peptide
thereof partly defective in the both terminals has an action to
promote division of salivary gland cells in a detection test of
.sup.3H-thymidine uptake (see non-patent document 3).
[0006] However, it has not been reported that lacritin or a
fragment thereof (partial peptide) is involved in adhesion between
a cell and extracellular matrix adhesion.
[0007] On the other hand, it is known that polypeptide generally
having glutamine or glutamic acid on the N-terminal is sometimes
unstable in an aqueous solution. As a stabilization method,
pyro-modification and the like, namely, a method including
synthesis of pyroglutamic acid derivative and the like, are known.
However, pyro-modification and the like has been reported to impair
inherent activity (patent document 3). [0008] patent document 1:
WO02/065943 [0009] patent document 2: WO05/119899 [0010] patent
document 3: JP-A-2003-528112 [0011] non-patent document 1: Frisch,
S. M. et al., Journal of Cell Biology 124, pp. 619-626 (1994)
[0012] non-patent document 2: Sanghi, S. et al., Journal of
Molecular Biology 310, pp. 127-139 (2001) [0013] non-patent
document 3: Wang, J. et al. Journal of Cell Biology 174, pp.
689-700 (2006)
SUMMARY OF INVENTION
Technical Problem
[0014] An object of the present invention is to provide a substance
capable of promoting adhesion between a cell and an extracellular
matrix, particularly a corneal epithelial cell and an extracellular
matrix, and superior in the stability in aqueous solutions.
Solution to Problem
[0015] The present inventors have conducted intensive studies in
view of the above-mentioned problems, and found that a polypeptide
having a particular partial sequence of lacritin can promote
adhesion between a corneal epithelial cell and an extracellular
matrix, and can further promote secretion of a tear protein from
lacrimal gland acinar cells. Furthermore, the present inventors
have succeeded in markedly improving the stability of the
polypeptide in aqueous solutions while maintaining a cell adhesion
promoting effect by modifying (e.g., pyro-modification) glutamine
on the N-terminal of the polypeptide, which resulted in the
completion of the present invention.
[0016] Accordingly, the present invention is as follows.
[1] A polypeptide comprising the amino acid sequence of SEQ ID NO:
1. [2] An agent for promoting cell adhesion, comprising the
polypeptide of the above-mentioned [1]. [3] A method of promoting
cell adhesion, comprising contacting an effective concentration of
the polypeptide of the above-mentioned [1] with a cell. [4] A
medicament comprising the polypeptide of the above-mentioned [1] as
an effective ingredient. [5] The medicament of the above-mentioned
[4], which is aqueous liquid. [6] The medicament of the
above-mentioned [4], which is used for the prophylaxis or treatment
of corneal epithelium disorder. [7] A method for the prophylaxis or
treatment of corneal epithelium disorder in a patient in need
thereof, which comprises administering an effective amount of the
polypeptide of the above-mentioned [1]. [8] A polypeptide
comprising the amino acid sequence of SEQ ID NO: 1 for promoting
cell adhesion. [9] A polypeptide comprising the amino acid sequence
of SEQ ID NO: 1 for the prophylaxis or treatment of corneal
epithelium disorder.
[0017] While a preferable embodiment of the present invention is
shown in the following, it should be appreciated that those of
ordinary skill in the art can appropriately practice the embodiment
and the like of the present invention from the explanation thereof
and attached drawings, as well as conventional technique well known
in the art, and readily understand the action and effect afforded
by the present invention.
Advantageous Effects of Invention
[0018] The present invention can provide a novel polypeptide stable
in aqueous solutions, which is capable of promoting adhesion
between a cell and an extracellular matrix, particularly a corneal
epithelial cell and an extracellular matrix. With such novel
polypeptide, a medicament superior in preservation stability,
particularly such medicament in the form of an aqueous liquid, can
be provided.
[0019] A corneal epithelial sheet, which functions stably for a
long time by preventing cell dropout, can be prepared by adding the
polypeptide of the present invention into a culture medium for
preparation of a corneal epithelial sheet for transplantation.
Furthermore, the present invention can provide a medicament useful
for the prophylaxis or treatment of corneal epithelium
disorder.
BRIEF DESCRIPTION OF DRAWINGS
[0020] FIG. 1 shows an influence of pyro-modification on the
stability of partial peptide of lacritin in aqueous solution.
[0021] FIG. 2 is a graph showing the results of cell adhesion
promoting effect of various partial peptides of lacritin, wherein
the vertical axis shows the cell adhesion rate against that without
addition of polypeptide as 100%, and the horizontal axis shows the
kind of peptide.
[0022] FIG. 3 is a graph showing the results of the promoting
effect of partial peptides of lacritin on tear protein
(lactoferrin) secretion from monkey lacrimal gland acinar cells,
wherein the vertical axis shows the amount of lactoferrin secreted
in the medium against PBS as 100%, and the horizontal axis shows
evaluated various peptides.
DESCRIPTION OF EMBODIMENTS
[0023] The present invention is explained below. Unless
particularly specified, the terms in the present specification are
generally used to mean the same as in the pertinent field.
[0024] The polypeptide of the present invention is a polypeptide
comprising the amino acid sequence shown by the following SEQ ID
NO: 1.
Pyr Gly Thr Ala Lys Val Thr Ser Ser Arg Gln Glu Leu Asn Pro Leu Lys
Ser Ile Val Glu Lys Ser Ile Leu Leu Thr Glu Gln Ala Leu Ala Lys
Ala
(SEQ ID NO: 1)
[0025] wherein "Pyr" means pyroglutamic acid.
[0026] The amino acid sequence of SEQ ID NO: 1 corresponds to the
69th-102nd of human-derived full-length lacritin (see GenBank/EBI
data bank accession Nos. NM.sub.--033277 and ay005150 (genomic);
AAG32949 (extracellular glycoprotein lacritin precursor)) of SEQ ID
NO: 5 consisting of 138 residues, wherein the N-terminal (i.e.,
69th) glutamine is pyro-modified.
[0027] In the present invention, moreover, polypeptide having amino
acid sequence shown by SEQ ID NO: 1 may contain acetylglutamine
instead of N-terminal pyroglutamic acid (SEQ ID NO: 6). That is, a
polypeptide wherein the N-terminal (i.e., 69th) glutamine in the
69th-102nd region of the human-derived full-length lacritin (see
GenBank/EBI data bank accession Nos. NM.sub.--033277 and ay005150
(genomic); AAG32949 (extracellular glycoprotein lacritin
precursor)) consisting of 138 residues, which is shown by SEQ ID
NO: 5, is acetylated is also provided by the present invention.
Ac-Gln Gly Thr Ala Lys Val Thr Ser Ser Arg Gln Glu Leu Asn Pro Leu
Lys Ser Ile Val Glu Lys Ser Ile Leu Leu Thr Glu Gln Ala Leu Ala Lys
Ala (SEQ ID NO: 6)
[0028] wherein "Ac-Gln" means acetylglutamine.
[0029] Therefore, in the present specification, polypeptide wherein
the N-terminal glutamine is modified by pyro-modification or
acetylation is also referred to as "partial peptide of lacritin of
the present invention".
[0030] Moreover, the partial peptide of lacritin of the present
invention may be a polypeptide containing an amino acid sequence
wherein 1-3, preferably 1-2, more preferably 1, amino acid in the
amino acid sequence shown by SEQ ID NO: 1 and/or 6 may be deleted,
substituted or added, as long as an activity equivalent to that of
polypeptide having the aforementioned amino acid sequence shown by
SEQ ID NO: 1 and/or polypeptide having the amino acid sequence
shown by the aforementioned SEQ ID NO: 6, namely, the cell adhesion
promoting action of the polypeptide and stability in aqueous
solution, is maintained. However, the above-mentioned deletion,
substitution or addition of the amino acid is applied to the amino
acid other than the N-terminal, since the present invention is
characterized by the modification (e.g., pyro-modification,
acetylation) of the N-terminal glutamine.
[0031] In the present specification, the "amino acid" generally
means "natural amino acid". However, it may be "non-natural amino
acid" as long as it satisfies the object of the present invention.
Here, the "natural amino acid" means an L-isomer of natural amino
acid. The natural amino acid includes glycine, alanine, valine,
leucine, isoleucine, serine, methionine, threonine, phenylalanine,
tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid,
asparagine, glutamic acid, glutamine, arginine, ornithine and
lysine. Unless otherwise specified, all amino acids in the present
specification are L-forms. However, an embodiment using amino acid
in a D-form is also within the scope of the present invention.
Here, the "non-natural amino acid" means amino acid generally
absent in a protein. Examples of the non-natural amino acid include
D-form of norleucine, para-nitrophenylalanine, homophenylalanine,
para-fluorophenylalanine, 3-amino-2-benzylpropionic acid or
homoarginine and D-phenylalanine, or other modified amino acid.
[0032] In the present specification, the "deletion of amino acid"
means removal of constituent amino acid from any position of the
amino acid sequence.
[0033] In the present specification, the "substitution of amino
acid" means substitution of the constituent amino acid with other
amino acid at any position of the amino acid sequence. As the
substitution of the amino acid, conservative substitution is
preferable. The conservative substitution means a substitution such
that amino acid is substituted by other amino acid having similar
property, due to which those of ordinary skill in the art in the
peptide chemistry expect the secondary structure and hydropathy
property of polypeptide do not change substantially. As groups of
amino acids in conservative substitution with each other, the
following are generally known: (1) glycine, asparagine, glutamine,
cysteine, serine, threonine and tyrosine; (2) alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine and
tryptophan; (3) glycine, alanine, serine, threonine and methionine;
(4) leucine, isoleucine and valine; (5) glutamine and asparagine;
(6) glutamic acid and aspartic acid; (7) arginine, lysine and
histidine; (8) phenylalanine, tryptophan and tyrosine.
[0034] The substitution of the amino acid may be that to modified
amino acid. As the modified amino acid, amino acid that underwent
addition of amino-protecting group (e.g., acetylation, formylation,
butyloxycarbonylation (Boc-modification),
fluorenylmethoxycarbonylation (Fmoc-modification), esterification
of carboxyl group (ethylation and the like) and the like can be
mentioned.
[0035] In the present specification, the "addition of amino acid"
means addition of any amino acid to any position of an amino acid
sequence, and includes insertion of an amino acid.
[0036] In the present specification, the "peptide" is a generic
term of a substance having a peptide bond formed by dehydration of
two or more of the above-mentioned amino acids (natural,
non-natural amino acids) between amino group and carboxyl group.
When the number of the bonded amino acids is 2, it is dipeptide,
when the number is 3, it is tripeptide, when several to about dozen
amino acids are bonded, it is oligopeptide, and a more number of
amino acids are bonded, it is polypeptide.
[0037] In the present specification, the "partial peptide of
lacritin" means a polypeptide consisting of a part of the amino
acid sequence of lacritin shown by SEQ ID NO: 5.
[0038] As mentioned above, the polypeptide of the present invention
may have an amino acid sequence wherein 1-3, preferably 1-2, more
preferably 1, amino acid may be deleted, substituted or added, as
long as it has an activity equivalent to that of polypeptide having
the aforementioned amino acid sequence shown by SEQ ID NO: 1 and/or
polypeptide having the amino acid sequence shown by the
aforementioned SEQ ID NO: 6, and such polypeptide is also within
the present invention. Here, "having an activity equivalent" means
the presence of not less than about 80%, preferably not less than
about 90%, of a cell adhesion promoting action of the polypeptide
before deletion, substitution or addition of the amino acid, as
well as the presence of not less than about 80%, preferably not
less than about 90%, of the stability of the polypeptide before
deletion, substitution or addition of the amino acid in aqueous
solution. In the present specification, the "cell adhesion
promoting action" is an action to promote adhesion between a cell
and extracellular matrix (cell--substrate adhesion) or adhesion
between cells, preferably an action to promote adhesion between a
cell and extracellular matrix. Here, for example, when promoting
cell--substrate adhesion, to "promote cell adhesion" corresponds to
increasing the number of cells that have adhered to the substrate
by the presence of a test polypeptide as compared to the absence
thereof. Specifically, this action can be assayed as described in
the below-mentioned Examples by adding a test polypeptide onto a
plate coated with a suitable extracellular matrix, forming a layer
of corneal epithelial cells thereon, incubating the cells for a
given time, and counting the adhered cells. As the stability in
aqueous solution, stability in an aqueous solution such as
phosphate buffered saline (PBS) and the like can be exemplified.
This action can be evaluated by adding test polypeptide to a
suitable aqueous solution such as PBS and the like and, after
preservation under given conditions, measuring the residual ratio
of polypeptide by HPLC and the like, as described in the
below-mentioned Examples.
[0039] As the superior stability of the partial peptide of lacritin
of the present invention in aqueous solution, specifically,
possible preservation for at least 4 weeks at 4.degree.
C.-40.degree. C., or possible preservation for at least 2 weeks at
60.degree. C. can be mentioned.
[0040] The polypeptide of the present invention may be in the form
of a salt according to a known method. As the salt of the
polypeptide, a pharmacologically acceptable salt with a base (e.g.,
alkali metal) or a salt with an acid, and a pharmacologically
acceptable acid addition salt is particularly preferable. Examples
of the pharmacologically acceptable acid addition salt include
salts with inorganic acids (e.g., hydrochloric acid, phosphoric
acid, hydrobromic acid, sulfuric acid), salts with organic acids
(e.g., acetic acid, formic acid, propionic acid, fumaric acid,
maleic acid, succinic acid, tartaric acid, citric acid, malic acid,
oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic
acid) and the like.
[0041] The polypeptide of the present invention can be produced by
a conventional chemical synthesis method, a recombination DNA
technique or the like.
[0042] When the polypeptide of the present invention is produced by
a chemical synthesis method, it can be produced according to a
known peptide synthesis method. Examples of the peptide synthesis
method include solid phase synthesis process, liquid phase
synthesis process and the like, with preference given to solid
phase synthesis process. Example of the solid phase synthesis
process includes Fmoc method. The Fmoc method is a method of
protecting an .alpha.-amino group with a 9-fluorenylmethoxycarbonyl
(Fmoc) group, and protecting a side chain functional group with a
t-butyl alcohol protecting group, wherein an Fmoc-amino acid is
condensed while deprotecting an Fmoc group with piperidine, which
is a secondary amine, and the side chain protecting group is
finally removed by weak acid such as trifluoroacetic acid. That is,
a series of operations of selective removal of
.alpha.-amino-protecting group and condensation of the protected
amino acid is repeated from the C-terminal side of the peptide to
be synthesized to construct a protected peptide chain, and the
protecting group of the side chain functional group is removed to
give the object peptide.
[0043] In the solid phase peptide synthesis method, synthesis by an
automatic peptide synthesis apparatus is also generally used (e.g.,
"Shin-Seikagaku Jikken-Koza vol. 1, protein IV" (1992) edited by
the Japan Biochemical Learned Society, Tokyo Kagaku Dojin; "The
Peptides: Analysis, Synthesis, Biology" Vol. 1-5, ed. by E. Gross,
J. Meienhofer; Vol. 6-9, ed. by S. Udenfriend, J. Meienhofer,
Academic Press, New York (1979-1987)).
[0044] When the polypeptide of the present invention is produced by
a recombinant DNA technique, for example, a partial peptide of
lacritin wherein N-terminal is glutamine is prepared by a
recombinant DNA technique and then the N-terminal glutamine is
pyro-modified to obtain the polypeptide of the present invention.
Firstly, primers are designed based on the base sequence of cDNA
encoding the partial peptide of lacritin wherein N-terminal is
glutamin and, using a suitable cDNA library as a template, the
object sequence is amplified by polymerase chain reaction (PCR),
whereby a cDNA encoding the polypeptide can be produced. Such PCR
method is well known in the pertinent technical field and
described, for example, in "PCR Protocols, A Guide to Methods and
Applications", Academic Press, Michael, et al., eds., 1990. Then, a
DNA encoding the polypeptide is incorporated into a suitable
expression vector, which is then introduced into either eucaryote
or prokaryote, and each chain is expressed to give the desired
polypeptide. Examples of the host cell usable for expression of the
polypeptide include, but are not limited to, prokaryote hosts such
as Escherichia coli, Bacillus subtilis and the like, and eucaryote
hosts such as yeast, fungi, insect cell, mammalian cell and the
like. Vector is a single strand or double stranded nucleic acid
molecule that can be transfected into a cell and is replicatable in
the cell genome or independently of the cell genome. An expression
vector contains a promoter region that drives DNA expression, and
may further contain a transcription and translation regulating
sequence, for example, TATA box, capping sequence; CAAT sequence,
3' non-coding region, enhancer and the like. Examples of the
promoter to be used in a prokaryote host include bla promoter, cat
promoter and lacZ promoter, and that to be used in an eucaryote
host include promoter of mouse metallothionein I gene sequence,
herpes virus TK promoter, SV40 early promoter, yeast glycolytic
enzyme gene sequence promoter and the like. Examples of the vector
include, but are not limited to, pBR322, pUC118, pUC119,
.lamda.gt10, .lamda.gt11, pMAM-neo, pKRC, BPV, vaccinia, SV40,
2-micron and the like.
[0045] Expression vector preferably contains one or more markers so
that a host cell containing the vector can be selected. As a
marker, those affording nutrition to a complementing auxotrophic
host, antibiotic resistance (e.g., ampicillin, tetracycline,
neomycin, hygromycin, geneticin etc.) or heavy metal resistance
(e.g., copper) can be used.
[0046] Furthermore, a vector can be constructed such that the
polypeptide is secreted and expressed using a signal sequence or
the polypeptide of the present invention is expressed in the form
of a fusion polypeptide with different polypeptide. Using a fusion
polypeptide, the stability of the polypeptide can be improved or
purification can be facilitated. Construction of such an expression
vector is well known in the pertinent technical field.
[0047] A vector constructed to express the polypeptide can be
introduced into a suitable host cell by transformation,
transfection, conjugation, protoplast fusion, electroporation,
particle gun technique, calcium phosphate precipitation, direct
microinjection and the like. The polypeptide can be obtained by
growing a cell containing the vector in a suitable medium to
produce the polypeptide, recovering a desired recombinant
polypeptide from the cell or medium, and purifying the
polypeptide.
[0048] A polypeptide having a modified glutamine present in the
N-terminal may be synthesized using modified glutamine as a
starting material by chemical synthesis and the like, and also may
be synthesized by modification of glutamine present in the
N-terminal of the polypeptide without N-terminal modification,
which is produced as mentioned above. For example, for
pyro-modification of glutamine present in the N-terminal,
cyclization by intramolecular condensation of amino group of
glutamine and free .gamma.-carboxylic acid functional group it has
may be performed. Examples of the method for pyro-modification
include a method comprising heating polypeptide, a method
comprising use of enzyme, a method comprising changing the
composition of buffer, a method comprising adding salt and the like
(Biotechnology and Bioengineering, Vol. 97, No. 3, Jun. 15, 2007).
For acetylation of glutamine present in the N-terminal, a method
comprising use of acetic anhydride and the like can be used
(Molecular Immunology 40 (2003) 943-948; J. Peptide Res. 2001, 57,
528-538).
[0049] A partial peptide of lacritin that underwent
pyro-modification by any method may be used in the present
ingention. Similarly, a partial peptide of lacritin that underwent
acetylation by any method may be used in the present ingention.
[0050] The polypeptide of the present invention also includes an
amino acid sequence shown by SEQ ID NO: 1 and/or SEQ ID NO: 6,
wherein 1-3, preferably 1-2, more preferably 1, amino acid may be
deleted, substituted or added, and maintaining an activity
equivalent to that of polypeptide having the aforementioned amino
acid sequence shown by SEQ ID NO: 1 and/or SEQ ID NO: 6, namely,
the cell adhesion promoting action of the polypeptide and stability
in aqueous solution. Said polypeptide can also be produced by
obtaining a cDNA encoding the polypeptide by entering the
corresponding modifications into the amino acid positions deleted,
substituted or added by a known means such as Kunkel method, Gapped
duplex method and the like or a method analogous thereto, and
subjecting the gene to a recombinant DNA technique similar to those
mentioned above. A mutation can be introduced into the gene by, for
example, using a mutation introduction kit based on a site-specific
mutation induction method (e.g., Mutant-K (Takara Bio Inc.),
Mutant-G (Takara Bio Inc.)) and the like or LA PCR in vitro
Mutagenesis series kit of Takara Bio Inc.
[0051] The polypeptide of the present invention obtained as
mentioned above can be isolated and purified by a known method.
Examples of known isolation and purification methods include
salting out, solvent precipitation, dialysis, ultrafiltration, gel
filtration, SDS-polyacrylamide gel electrophoresis, ion exchange
chromatography, affinity-chromatography, reversed-phase high
performance liquid chromatography, isoelectric focusing and the
like.
[0052] Each peptide used may be commercially available, or
specifically, synthesized by contractors (e.g., Bachem, Biologica
Co., Biosynthesis Inc. etc.).
[0053] The thus-obtained polypeptide of the present invention
promotes adhesion between a cell and an extracellular matrix,
particularly a corneal epithelial cell and an extracellular matrix.
The following explains its specific use.
(1) Culture Medium Containing the Polypeptide of the Present
Invention for Preparation of Corneal Epithelial Sheet
[0054] The polypeptide of the present invention can particularly
promote adhesion between a corneal epithelial cell and a base
material during preparation of a corneal epithelial sheet for
transplantation, due to the promoting effect on the adhesion
between a corneal epithelial cell and an extracellular matrix.
[0055] The corneal epithelial sheet is a substitute for living
cornea and used for the treatment of cornea opacity to recover
visual acuity and the like. It is used for the treatment of a
refractory cornea epithelial disease such as stevens-johnson
syndrome, chemical trauma and the like. A corneal epithelial sheet
is prepared by, for example, adding a cell such as a corneal
epithelial cell and the like on a base material such as amniotic
membrane, collagen sheet and the like in a serum-containing medium,
culturing the cell, and layering by coculture with 3T3 fibroblast,
air-lifting and the like (Ophthalmology, vol. 42, No. 3, pages
245-250, 2000). As a method for preparation of a corneal epithelial
sheet, known methods described in WO03/043542, JP-A-2004-298447,
JP-A-2004-261533, JP-A-2002-331025 and the like are applied.
[0056] In the production method of the corneal epithelial sheet, a
known base material used for the production of a corneal epithelial
sheet can be used, and any of a base material derived from a living
organism and an artificially prepared base material can be used.
Specifically, as a base material derived from a living organism,
amniotic membrane can be mentioned, and as an artificial base
material, a collagen sheet can be mentioned. The amniotic membrane
covers the uterus and the outermost layer of the placenta, and is
discharged from the body with the placenta during childbirth.
[0057] As a culture medium to be used for the cell culture, known
culture media used for the production of a corneal epithelial
sheet, such as EpiLife medium (manufactured by Cascade Biologics
Inc.), DMEM/F12 medium (manufactured by Invitrogen Corporation),
DMEM medium (manufactured by Invitrogen Corporation) and the like
can be used, and the culture medium can contain a known serum.
While the culture temperature is not particularly limited as long
as the above-mentioned cells can grow well, it is generally about
15.degree. C.-45.degree. C. While the culture time is not
particularly limited as long as the above-mentioned cell can grow
well, it is generally about 1-30 days.
[0058] The polypeptide of the present invention added to a culture
medium for the preparation of the corneal epithelial sheet can be
an active ingredient for promoting adhesion between a corneal
epithelial cell and a base material. The concentration of
polypeptide in a culture medium is generally 0.0001 w/v %-0.1 w/v
%, preferably 0.001 w/v %-0.01 w/v %. The polypeptide of the
present invention promotes fixation between an extracellular matrix
of the base material and a corneal epithelial cell, and enables
preparation of a robust corneal epithelial sheet, which functions
stably for a long time by preventing cell dropout.
(2) Medicament Containing Polypeptide of the Present Invention
[0059] The polypeptide of the present invention is useful as an
agent for promoting cell adhesion, since it has an action to
promote cell adhesion, particularly adhesion between a cell and
extracellular matrix. In the present specification, the "an agent
for promoting cell adhesion" means a substance having the
above-mentioned "cell adhesion promoting action", particularly a
substance that promotes adhesion between a cell and extracellular
matrix. Here, the level of promotion of cell adhesion when, for
example, it is an agent to promote adhesion between a cell and
extracellular matrix, is such that addition of polypeptide of the
present invention increases the number of cells that significantly
adhere to the substrate. The agent for promoting adhesion of the
present invention is used for a cell (e.g., corneal epithelial
cell, corneal endothelium cell, conjunctival cell and the like)
derived from a mammal (e.g., rat, mouse, guinea pig, bird, sheep,
horse, bovine, swine, monkey, chimpanzee, human etc.), preferably a
corneal epithelial cell derived from human. The extracellular
matrix is not particularly limited as long as it can adhere to a
cell and includes (1) fibrous protein such as collagen, elastin and
the like, (2) cell adhesion glycoprotein such as fibronectin,
laminin, vitronectin and the like, (3) glycoconjugate such as
glycosaminoglycans including heparin, hyaluronic acid, chondroitin
sulfate and the like, and the like, as well as basal lamina (e.g.,
Bowman's membrane, Descemet's membrane, amniotic membrane and the
like) comprised of these extracellular matrices.
[0060] An agent for promoting cell adhesion containing polypeptide
of the present invention is intended for application to various
clinical medicaments due to its cell adhesion promoting action as
mentioned later. Moreover, its use as a research reagent is also
provided by the present invention. An agent for promoting cell
adhesion containing polypeptide of the present invention can be
used as a reagent for researches for the study of extracellular
matrix, study of cell signal transduction involving adhesion and
the like.
[0061] It has been reported that cell adhesion to an extracellular
matrix is important for cell survival (Frisch, S. M. et al., J.
Cell Biol. 1994, 124, 619., Porcu, M., et al., Cornea 2007, 26,
73.). In the cornea, inhibition of cell adhesion due to the
disappearance of laminin 5, which is one of the extracellular
matrices, has also been reported to enhance death of corneal
epithelial cells. Since the polypeptide of the present invention
promotes adhesion of a corneal epithelial cell to the basal lamina
(Bowman's membrane and the like) of corneal epithelium comprised of
the extracellular matrix, it suppresses death of corneal epithelial
cells on the surface layer of the eyes. Moreover, it is known that
cell motilities consisting of division, migration (extension) and
adhesion are involved in the repair of the corneal epithelium
(Suzuki, K. et al., Prog. Retin. Eye Res. 2003, 22, 113). The
polypeptide of the present invention promotes adhesion process in
the cell motility, thereby promoting repair of corneal epithelial
injury (i.e., wound or defect).
[0062] Therefore, a medicament containing the polypeptide of the
present invention is useful for the treatment of a corneal
epithelial disorder. As a specific disease causing a corneal
epithelial disorder, keratitis due to physical or chemical
stimulation, allergy, bacterial or fungal or virus infection and
the like, corneal ulcer, corneal epithelial detachment (corneal
erosion), corneal epithelial edema, corneal burn, cornea corrosion
due to chemical substance and the like, dry eye, xerophthalmia,
chronic superficial keratitis, superficial punctate keratopathy,
corneal epithelial erosion, persistent corneal epithelial defects
and the like can be mentioned, and is not particularly limited. The
polypeptide of the present invention is particularly useful for the
treatment of a corneal epithelial disorder associated with these
diseases.
[0063] In addition, the polypeptide of the present invention has a
promoting action of tear fluid secretion from lacrimal gland acinar
cells. Tear fluid covers eyeball surface composing of cornea and
conjunctiva, maintains wettability of the keratoconjunctiva, and
prevents drying. In recent years, however, an increasing number of
people complain about various symptoms such as tired feeling,
feeling of foreign substance, i.e., dry eye syndrome, due to dried
cornea-conjunctiva surface associated with tear fluid decreases,
dry eye during wearing contact lenses, or dry eye during operation
of office automation equipment and the like. Dry eye sometimes
accompany corneal epithelial disorder, corneal epithelial erosion
and the like due to disorder of corneal epithelial cells and, in
serious cases, may develop corneal ulcer or ophthalmic infection.
To alleviate such various symptoms associated with drying, an
artificial tear fluid mainly containing salts such as sodium
chloride and the like, and eye drops containing
hydroxyethylcellulose, chondroitin sulfate or hyaluronic acid and
the like are used. As the situation stands, however, a satisfactory
agent has not been developed as yet. Although such symptomatic
therapy can alleviate symptoms, it is not a causal therapy for a
basic treatment. Tear fluid is considered to have, based on its
inherent function, a therapeutic effect on keratoconjunctival
disorders due to dry eye. Therefore, a substance that directly acts
on the lacrimal gland to promote tear fluid secretion is expected
to be a useful prophylactic or therapeutic drug for dry eye and
diseases associated with dry eye.
[0064] While the dosage form of a pharmaceutical product containing
the polypeptide of the present invention is not particularly
limited, preferred are eye drops, eye washes, eye ointment, tablet
and the like. More preferred is the form of an aqueous liquid
(especially, eye drops) since it has significantly improved
stability in aqueous solution. Here, the "aqueous liquid" means
liquid with aqueous solvent. Examples of the aqueous solvent
include, but are not limited to, distilled water for injection,
purified water purified by ion-exchange resin or distillation,
sterile purified water which is prepared by sterilizing the
purified water, and the like. It is also preferable that the
pharmaceutical product containing the polypeptide of the present
invention is provided as a form of preparation to be dissolved when
in use, which is reservable at room temperature after dissolution.
These can be prepared by using a technique widely used. For
example, eye drops can be prepared by appropriately blending
additives such as isotonicity agent, buffering agent, pH adjuster,
solubilizer, thickener, stabilizer, preservative and the like. In
addition, stable eye drops can also be obtained by adding pH
adjuster, thickener, dispersing agent and the like and suspending a
drug.
[0065] Examples of the isotonicity agent include, but are not
limited to, glycerol, propylene glycol, sodium chloride, potassium
chloride, sorbitol, mannitol, glucose, boric acid and the like.
[0066] Examples of the buffering agent include, but are not limited
to, phosphoric acid, phosphate, citric acid, acetic acid,
.epsilon.-aminocaproic acid, tromethamol, citrate, acetate, boric
acid, glutamine, carbonate and the like.
[0067] Examples of the pH adjuster include, but are not limited to,
hydrochloric acid, citric acid, phosphoric acid, acetic acid,
sodium hydroxide, potassium hydroxide, boric acid, borax, sodium
carbonate, sodium hydrogen carbonate and the like.
[0068] Examples of the solubilizer include, but are not limited to,
polysorbate 80, polyoxyethylene hydrogenated castor oil 60,
macrogol 4000 and the like.
[0069] Examples of the thickener and dispersing agent include, but
are not limited to, cellulose polymers such as
hydroxypropylmethylcellulose, hydroxypropylcellulose and the like,
polyvinyl alcohol, polyvinylpyrrolidone and the like. Moreover,
examples of the stabilizer include edetic acid, sodium edetate and
the like.
[0070] Examples of the conventional preservative include, but are
not limited to, sorbic acid, potassium sorbate, benzalkonium
chloride, benzethonium chloride, methyl p-hydroxybenzoate, propyl
p-hydroxybenzoate, chlorobutanol, boric acid, sodium edetate and
the like. These preservatives may also be used in combination.
[0071] Eye drops containing the polypeptide of the present
invention desirably has a pH of 4-8, and an osmotic pressure ratio
of around 1.
[0072] In a pharmaceutical product containing the polypeptide of
the present invention, the concentration of the polypeptide of the
present invention can be set according to the symptom, age and the
like and is not particularly limited. For example, when the
polypeptide of the present invention is contained in eye drops, eye
washes and the like, it is about 0.00003 w/v %-about 5 w/v %,
preferably about 0.00001 w/v %-about 0.5 w/v %, about 0.0001 w/v
%-about 0.0005 w/v %, about 0.001 w/v %-about 0.005 w/v %, about
0.01 w/v %-about 0.05 w/v %, more preferably about 0.003 w/v
%-about 0.5 w/v %, most preferably about 0.001 w/v %-about 0.5 w/v
%. The dose in the case of eye drops is, for example, one drop to
several drops per ocular instillation, which is given once to
several times per day. Eye drops may be a general ophthalmic
solution, or an ophthalmic solution to be dissolved when in
use.
[0073] A pharmaceutical product containing the polypeptide of the
present invention as an active ingredient can be used for, for
example, mammals (e.g., rat, mouse, guinea pig, bird, sheep, horse,
bovine, swine, monkey, chimpanzee, human etc.) and the like.
EXAMPLES
[0074] The present invention is explained in detail in the
following by referring to Examples, which are not to be construed
as limitative.
Example 1
Synthesis of Polypeptide 1 (Pyr Lac50-83)
[0075] Polypeptide 1 was synthesized by a solid phase synthesis
process. To be specific, a fluorenylmethoxycarbonyl (Fmoc) group
was introduced into amino acid and the amino acid was supported by
a resin. Then, an amide bond formation reaction was performed using
dichloromethane as a solvent, and
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium (HBTU) and
N-methylpyrrolidone (NMP) as coupling reagents. The protecting
group was eliminated by using DMF/20% piperidine. The obtained
product was purified by high performance liquid chromatography
(column: ODS, solvent: water/acetonitrile/0.05% TFA). As a result,
polypeptide 1 of the following amino acid sequence was
obtained.
Polypeptide 1: Pyr Gly Thr Ala Lys Val Thr Ser Ser Arg Gln Glu Leu
Asn Pro Leu Lys Ser Ile Val Glu Lys Ser Ile Leu Leu Thr Glu Gln Ala
Leu Ala Lys Ala (SEQ ID NO: 1)
[0076] wherein "Pyr" means pyroglutamic acid. Bachem (manufacturer
and supplier): Product No. 4064390 white powder MALDI-TOF-MS
Calcd.: 3636.20; Found: 3636.89;
Purity (HPLC A/A %)>92%
Comparative Example 1
Synthesis of Polypeptide 2 (Lac50-83)
[0077] In the same manner as in Example 1, polypeptide 2 of the
following amino acid sequence was obtained.
polypeptide 2: Gln Gly Thr Ala Lys Val Thr Ser Ser Arg Gln Glu Leu
Asn Pro Leu Lys Ser Ile Val Glu Lys Ser Ile Leu Leu Thr Glu Gln Ala
Leu Ala Lys Ala (SEQ ID NO: 2) Biosynthesis Inc. (manufacturer)
white powder MALDI-TOF-MS Calcd.: 3653.27; Found: 3652.28;
Purity (HPLC A/A %) 99.26%
Comparative Example 2
Synthesis of Polypeptide 3 (Pyr Lac50-94)
[0078] In the same manner as in Example 1, polypeptide 3 of the
following amino acid sequence was obtained.
polypeptide 3: Pyr Gly Thr Ala Lys Val Thr Ser Ser Arg Gln Glu Leu
Asn Pro Leu Lys Ser Ile Val Glu Lys Ser Ile Leu Leu Thr Glu Gln Ala
Leu Ala Lys Ala Gly Lys Gly Met His Gly Gly Val Pro Gly Gly (SEQ ID
NO: 3) wherein "Pyr" means pyroglutamic acid. Bachem (manufacturer
and supplier): Product No. 4064388 white powder MALDI-TOF-MS
Calcd.: 4571.28; Found: 4571.40;
Purity (HPLC A/A %): >90%
Comparative Example 3
Synthesis of Polypeptide 4 (Lac50-94)
[0079] In the same manner as in Example 1, polypeptide 4 of the
following amino acid sequence was obtained.
polypeptide 4: Gln Gly Thr Ala Lys Val Thr Ser Ser Arg Gln Glu Leu
Asn Pro Leu Lys Ser Ile Val Glu Lys Ser Ile Leu Leu Thr Glu Gln Ala
Leu Ala Lys Ala Gly Lys Gly Met His Gly Gly Val Pro Gly Gly (SEQ ID
NO: 4) Biosynthesis Inc. (manufacturer) white powder MALDI-TOF-MS
Calcd.: 4588.35; Found: 4590.14;
Purity (HPLC A/A %) 96.88%
Comparative Example 4
Synthesis of Polypeptide 5 (Lac49-83)
[0080] In the same manner as in Example 1, polypeptide 5 of the
following amino acid sequence was obtained.
polypeptide 5: Val Gln Gly Thr Ala Lys Val Thr Ser Ser Arg Gln Glu
Leu Asn Pro Leu Lys Ser Ile Val Glu Lys Ser Ile Leu Leu Thr Glu Gln
Ala Leu Ala Lys Ala (SEQ ID NO: 9) Biosynthesis Inc. (manufacturer)
white powder MALDI-TOF-MS Calcd.: 3752.4; Found: 3751.38;
Purity (HPLC A/A %) 96.13%
Experimental Example 1
Influence of Pyro-Modification on Stability of Partial Peptide of
Lacritin in Aqueous Solution
[0081] When the partial peptide of lacritin of the present
invention is produced as a medicament, particularly, a medicament
provided in the form of an aqueous liquid, stability thereof in
aqueous solution is desired. Therefore, an influence of
pyro-modification of N-terminal glutamine in the present invention
on the stability in aqueous solution was examined.
[0082] Using Pyr Lac50-83 produced in Example 1 and Lac50-83
produced in Comparative Example 1, and changing preservation
conditions in PBS (Phosphate-Buffered Salines, pH 7.2 (Invitrogen);
component (/100 mL): KH.sub.2PO.sub.4 0.021 g, NaH.sub.2PO.sub.4
0.0726 g, NaCl 0.9 g) variously, the residual ratio (%) of the
peptide content was measured. The concentration of each peptide was
10.sup.-4 M. Pyr Lac50-83 and Lac50-83 are polypeptides solely
different in the presence of pyro-modification (Pyr Lac50-83) or
absence of pyro-modification (Lac50-83), of N-terminal glutamine,
as is clear from the above-mentioned amino acid sequence.
[Preservation Conditions]
[0083] temperature: 4.degree. C., 25.degree. C., 40.degree. C.,
60.degree. C. period: 7 days, 14 days, 21 days, 28 days
[0084] Both polypeptides were preserved under respective
preservation conditions, peptide content was measured by HPLC
chromatography, and the residual ratio thereof was calculated. In
addition, the residual ratio was calculated by the following
formula 1, while taking into consideration as far as possible water
evaporation from the preservation container.
residual rate (after water correction) (%)=residual rate (%) before
water correction.times.(100-water permeation rate (%) of
container)/100 [formula 1]
wherein "the water permeation rate of container" is obtained by
measuring the weight of empty container (tare weight), and sample
weight before and after preservation, and calculating according to
the following formula 2. (wherein the sample weight is the total
weight of preservation container and solution)
water permeation rate (%) of container=(sample weight (g) before
preservation-sample weight (g) after preservation under each
condition)/(sample weight (g) before preservation-weight (g) of
empty preservation container).times.100 [formula 2]
[HPLC Chromatography Conditions]
[0085] detector: ultraviolet absorption spectrophotometer
(measurement wavelength: 220 nm)
[0086] column: commercially available column of a stainless tube
with inner diameter 4.6 mm, length 150 mm, which is filled with
liquid chromatography octadecylsilylated silica gel with average
particle size of 5 .mu.m was used.
(Capcel pak C18 UG120 .ANG., S5 .mu.m 4.6 mm.times.150 mm, Shiseido
Co., Ltd.) column temperature: constant temperature near 20.degree.
C. mobile phase A: trifluoroacetic acid solution (0.5.fwdarw.1000)
(solvent: purified water) mobile phase B: trifluoroacetic acid
solution (0.5.fwdarw.1000) (solvent: acetonitrile) solution sending
of mobile phase: the mixing ratio of mobile phase A and mobile
phase B was changed as follows to control linear density
gradient.
TABLE-US-00001 time (min) after mobile phase A mobile phase B
injection (vol %) (vol %) .sup. 0-2.5 90 10 2.5-27.5 90.fwdarw.35
10.fwdarw.65 27.5-30.sup. 35.fwdarw.10 65.fwdarw.90 30-35 10 90
35-40 10.fwdarw.90 90.fwdarw.10 40-50 90 10
flow: 0.5 mL/min Autosampler temperature: constant temperature near
4.degree. C. Injector washing: acetonitrile aqueous solution
(50.fwdarw.100) (solvent: purified water)
[0087] The results are shown in FIG. 1.
[0088] It was found that pyro-modification enables longer-term
preservation in aqueous solution, and preservation at a higher
temperature.
Experimental Example 2
Promoting Effect by Partial Peptide of Lacritin on Adhesion of
Human Corneal Epithelial Cell to Extracellular Matrix
[0089] In Experimental Example 1, pyro-modification of N-terminal
glutamine was found to markedly improve stability of polypeptide in
aqueous solution. Thus, whether or not the inherent property of
partial peptide of lacritin, i.e., cell adhesion promoting effect,
is influenced by pyro-modification was examined.
[0090] As a negative control, lacritin peptides (Lac24-56,
Lac50-65) having different sequence from that of the present
invention were used. These peptides were prepared in the same
manner as in the aforementioned peptide.
polypeptide Lac24-56
Glu Ile Ser Gly Pro Ala Glu Pro Ala Ser Pro Pro Glu Thr Thr Thr Thr
Ala Gln Glu Thr Ser Ala Ala Ala Val Gln Gly Thr Ala Lys Val Thr
(SEQ ID NO: 7)
[0091] Bachem (manufacturer and supplier): Product No. 4064384
polypeptide Lac50-65
Gln Gly Thr Ala Lys Val Thr Ser Ser Arg Gln Glu Leu Asn Pro Leu
(SEQ ID NO: 8)
[0092] Biosynthesis Inc. (manufacturer)
[0093] An extracellular matrix solution (10 .mu.g/mL, collagen type
IV: Becton, Dickinson and Company, catalog No. 354245) was added to
a 96 well plate (Iwaki Glass Company, Limited, catalog No.
3860-096). The solution was incubated at 37.degree. C. for 1 hr to
coat the plate with the extracellular matrix. After the redundant
extracellular matrix solution was removed, 0.1% BSA solution
(Sigma-Aldrich Co., catalog No. A3803) was added to block the
region not coated with the extracellular matrix. Successively, the
BSA solution was removed and the plate was washed twice with PBS,
and polypeptides 1-4 (concentration 100 .mu.g/mL) synthesized in
Example 1 (Pyr Lac50-83) and Comparative Examples 1-3 (Lac50-83,
Pyr Lac50-94, Lac50-94), and negative control polypeptide
(Lac24-56, Lac50-65) were added by 50 .mu.l, per well. Furthermore,
established human corneal epithelial cells (HCE-T: can be prepared
by the method described in Invest Ophthalmol Vis Sci. 1995, 36,
614) were cultured overnight under serum-free conditions and added
to the plate at 2.times.10.sup.4 cells/100 .mu.L DMEM/F12
medium/well. The plate was incubated at 37.degree. C. for 25 min to
allow adhesion of the cells to the plate.
[0094] Thereafter, the cells were fixed with 10% neutral buffered
formaldehyde liquid (NACALAI TESQUE, INC., catalog No. 37152-51),
and stained with 1% crystal violet staining solution (Sigma-Aldrich
Co., catalog No. C3886). The image of the stained cells was
uploaded using an inverted system microscope (Olympus Corporation,
IX71), and the area of the adhered cells was measured using
Image-Pro Plus Ver. 4.5 (Nippon Roper K.K.). The cell adhesion rate
obtained based on that without addition of polypeptide as 100% is
shown in FIG. 2.
[0095] As is clear from FIG. 2, polypeptide 1 (Pyr Lac50-83) was
confirmed to have an activity to promote cell adhesion between a
corneal epithelial cell and extracellular matrix, and confirmed to
be free of influence of pyro-modification of N-terminal glutamine
in Lac50-83. On the other hand, polypeptide 2 (Pyr Lac50-94) showed
lowered activity as compared to that of polypeptide before
pyro-modification (polypeptide 4: Lac50-94), and pyro-modification
of N-terminal glutamine of Lac50-94 was found to attenuate a cell
adhesion promoting effect.
[0096] From the above results, it was confirmed that the lacritin
partial polypeptide of the present invention is superior in that it
improves stability in aqueous solution while maintaining a cell
adhesion promoting effect.
Experimental Example 3
Promoting Effect by Partial Peptide of Lacritin on Secretion of
Tear Fluid Protein from Monkey Lacrimal Gland Acinar Cells
(Preparation of Acinar Cells)
[0097] The lacrimal glands of three monkeys (Macaca fascicularis)
were obtained from eve bio-science and used for the experiment. The
detail of the three monkeys used is as follows.
9 years 6 months (male, Vietnam) 10 years 9 months (male, China) 10
years 8 months (male, Vietnam)
[0098] The lacrimal gland thereof was minced in DMEM/F12
(Invitrogen Corporation, catalog No. 11330) containing 0.1 mg/mL
Trypsin inhibitor (Sigma, catalog No. T9003). Then, HBSS
(Invitrogen Corporation, catalog No. 14175) containing 0.76 mg/mL
EDTA (Wako Pure Chemical Industries, Ltd., catalog No. 345-01865)
was added to the lacrimal gland and the mixture was gently shaken
at 37.degree. C. for 7-12 min. Thereafter, the supernatant was
removed by centrifugation, DMEM/F12 medium containing 200 U/mL
collagenase A (Roche, catalog No. 11088785103), 698 U/mL
Hyarulonidase (Worthington, catalog No. LS002592), 10 U/mL DNase
(Roche, catalog No. 4536282) (CHD) was added, and the mixture was
gently shaken at 37.degree. C. for 10-40 min. The above-mentioned
step was performed twice. Thereafter, 20% FBS (Invitrogen
Corporation, catalog No. 10082-147) was added to quench the enzyme
reaction, and the cells were dispersed by a pipetting operation.
The residue was removed with 100 .mu.m (Becton, Dickinson and
Company, catalog No. 352360) and 40 .mu.m (Becton, Dickinson and
Company, catalog No. 352340) cell strainers. The cells were
separated by 10%, 30%, 60% Percoll (Sigma, catalog No. P4937) and
the cells that gathered between 30% and 60% were taken as acinar
cells.
(Detection of Tear Fluid Protein Lactoferrin Secreted in
Medium)
[0099] The monkey acinar cells prepared in this Experimental
Example were plated in a plate coated with rat collagen, Type I
(0.01 mg/cm.sup.2; BD Biosciences, catalog No. 354236), and
cultured in DMEM/F12 medium containing 10 ng/ml dexamethasone
(Sigma, catalog No. D2915), 1 mM putrescine (Sigma, catalog No.
P5780), 50 ng/ml EGF (Invitrogen Corporation, PHG0311), 25 .mu.g/ml
L-ascorbic acid (Sigma, catalog No. A4544), 1.times.
Insulin-transferrin-sodium selenite media supplement (Sigma,
catalog No. 11884), 10 .mu.g/mL Glutathione (Sigma, catalog No.
G6013) and 50 .mu.g/ml gentamicin (Invitrogen Corporation, catalog
No. 15750) for 1 day. The next day, preincubation was performed for
30 min in DMEM/F12 medium free of supplement. Then, DMEM/F12
containing partial peptide of lacritin was added, and the mixture
was incubated at 37.degree. C. for 10 min and the medium was
recovered.
[0100] As lacritin polypeptide, polypeptide 1 (Pyr Lac50-83) and
polypeptide 2 (Lac50-83) were used. As the control group, PBS was
used instead of polypeptide.
[0101] The collected medium was purified by ReadyPrep 2D clean up
kit (Bio-Rad Laboratories, Inc., catalog No. 163-2130), dissolved
in NuPAGE LDS sample buffer (Invitrogen Corporation, catalog No.
NP0007), and thermally denatured at 70.degree. C. for 10 min. An
equivalent amount of a sample was electrophoresed in 4-12% NuPAGE
Novex Bis Tris gel (Invitrogen Corporation, catalog No. NP0322BOX)
in MES buffer (Invitrogen Corporation, catalog No. NP0002) at 200 V
for 35 min, and blotted on a PVDF membrane (Nihon Millipore K.K.,
catalog No. IPVH00010) at 100 V for 60 min using Trans Blot Mini
Cell (Bio-Rad Laboratories, Inc.). The membrane was blocked with
TTBS (Bio-Rad Laboratories, Inc., catalog No. 170-6435) containing
0.5% skim-milk (Wako Pure Chemical Industries, catalog No.
198-10605) at room temperature for 30 min, and reacted overnight at
4.degree. C. with lactoferrin antibody (Sigma-Aldrich Co., catalog
No. L-3263) diluted 10000-fold. After washing the membrane with
TTBS, the membrane was reacted at room temperature for 60 min with
anti-rabbit HRP secondary antibody, which was diluted 10000-fold.
Then band(s) were detected and measured using ECL plus (GE
healthcare, catalog No. RPN2132).
[0102] The results are shown in FIG. 3. Three monkeys were used in
this Experimental Example, one sample was taken from each of the
first and second monkeys and two samples were taken from the third
monkey, 4 samples in total.
Experimental Example 4
Promoting Effect by Partial Peptide of Lacritin on Secretion of
Tear Fluid Protein from Rat and Rabbit Lacrimal Gland Acinar
Cells
(Preparation of Acinar Cells)
[0103] The lacrimal gland of rat and rabbit is minced in DMEM/F12
(Invitrogen Corporation, catalog No. 11330) containing 0.1 mg/mL
Trypsin inhibitor (Sigma, catalog No. T9003). Then, HBSS
(Invitrogen Corporation, catalog No. 14175) containing 0.76 mg/mL
EDTA (Wako Pure Chemical Industries, Ltd., catalog No. 345-01865)
is added and the mixture is gently shaken at 37.degree. C. for
15-20 min. Thereafter, the supernatant is removed by
centrifugation, DMEM/F12 medium containing 200 U/mL collagenase A
(Roche, catalog No. 11088785103), 698 U/mL Hyarulonidase
(Worthington, catalog No. LS002592), 10 U/mL DNase (Roche, catalog
No. 4536282) (CHD) is added, and the mixture is gently shaken at
37.degree. C. for 15-50 min. The above-mentioned step is performed
twice. Thereafter, 20% FBS (Invitrogen Corporation, catalog No.
10082-147) is added to quench the enzyme reaction, and the cells
are dispersed by a pipetting operation. The residue is removed with
100 .mu.m (Becton, Dickinson and Company, catalog No. 352360) and
40 .mu.m (Becton, Dickinson and Company, catalog No. 352340) cell
strainers. The cells are separated by 10%, 30%, 60% Percoll (Sigma,
catalog No. P4937) and the cells that gathered between 30% and 60%
are taken as acinar cells.
(Detection of Tear Fluid Protein Lactoferrin Secreted in
Medium)
[0104] The acinar cells of rat and rabbit are plated in a plate
coated with rat collagen, Type I (0.01 mg/cm.sup.2; BD Biosciences,
catalog No. 354236), and cultured in DMEM/F12 medium containing 10
ng/ml dexamethasone (Sigma, catalog No. D2915), 1 mM putrescine
(Sigma, catalog No. P5780), 50 ng/ml EGF (Invitrogen Corporation,
PHG0311), 25 .mu.g/ml L-ascorbic acid (Sigma, catalog No. A4544),
1.times. Insulin-transferrin-sodium selenite media supplement
(Sigma, catalog No. 11884), 10 .mu.g/mL Glutathione (Sigma, catalog
No. G6013) and 25 .mu.g/ml gentamicin (Invitrogen Corporation,
catalog No. 15750) for 1 day in a CO.sub.2 incubator. The next day,
preincubation is performed for 30 min in DMEM/F12 medium free of
supplement. Then, DMEM/F12 containing partial peptide of lacritin
is added, and the mixture is incubated at 37.degree. C. for 30 min
or 60 min and the medium is recovered.
[0105] As the amount of the tear fluid protein secreted in the
medium, peroxidase activity can be measured using Amplex Red
Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen Corporation,
catalog No. A22188), albumin amount can be measured using Rat
Albumin ELISA Quantitation Set (Bethyl Laboratories Inc., catalog
No. E100-125-12), and IgA amount can be measured using Rat IgA
ELISA Quantitation Set (Bethyl Laboratories Inc., catalog No.
E100-102).
Experimental Example 5
Promoting Effect by Partial Peptide of Lacritin on Secretion of
Tear Fluid Protein from Monkey Lacrimal Gland Acinar Cells in the
Presence of Cytokine
[0106] Cytokine has an action to decrease secretion of tear fluid
protein from acinar cells. In this Example, the presence or absence
of an effect to promote secretion of tear fluid protein by partial
peptide of lacritin in acinar cells showing decreased tear fluid
secretion function due to cytokine can be examined.
[0107] The lacrimal gland of monkey is minced in DMEM/F12
(Invitrogen Corporation, catalog No. 11330) containing 0.1 mg/mL
Trypsin inhibitor (Sigma, catalog No. T9003). Then, HBSS
(Invitrogen Corporation, catalog No. 14175) containing 0.76 mg/mL
EDTA (Sigma, catalog No. E-5134) is added and the mixture is gently
shaken at 37.degree. C. for 7-20 min. Thereafter, the supernatant
is removed by centrifugation, DMEM/F12 medium containing 200 U/mL
collagenase A (Roche, catalog No. 11088785103), 698 U/mL
Hyarulonidase (Worthington, catalog No. LS002592) and 10 U/mL DNase
(Roche, catalog No. 4536282) (CHD) is added, and the mixture is
gently shaken in an incubator at 37.degree. C. for 15-50 min. The
above-mentioned step is performed twice. Thereafter, 20% FBS
(Invitrogen Corporation, catalog No. 10082-147) is added to quench
the enzyme reaction, and the cells are dispersed by a pipetting
operation. The residue is removed with 100 .mu.m (Becton, Dickinson
and Company, catalog No. 352360) and 40 .mu.m (Becton, Dickinson
and Company, catalog No. 352340) cell strainers. The cells are
separated by 10%, 30%, 60% Percoll (Sigma, catalog No. P4937) and
the cells that gathered between 30% and 60% are taken as acinar
cells.
[0108] The acinar cells of monkey are plated in a plate coated with
rat collagen, Type I (0.01 mg/cm.sup.2; BD Biosciences, catalog No.
354236), and cultured in DMEM/F12 medium (cell culture medium)
containing 10 ng/ml dexamethasone (Sigma, catalog No. D2915), 1 mM
putrescine (Sigma, catalog No. P5780), 50 ng/ml EGF (Invitrogen
Corporation, PHG0311), 50 .mu.g/ml L-ascorbic acid (Sigma, catalog
No. A4544), 1.times. Insulin-transferrin-sodium selenite media
supplement (Sigma, catalog No. 11884), 10 .mu.g/mL Glutathione
(Sigma, catalog No. G6013) and 25 .mu.g/ml gentamicin (Invitrogen
Corporation, catalog No. 15750) for 1 day in a CO.sub.2 incubator.
The next day, the medium was changed to a cell culture medium
containing 10 ng/mL TNF-alpha (R&D Systems, catalog No. 210-TA)
and 10 ng/mL of IFN-gamma (R&D Systems, catalog No. 285-IF),
and the cells are cultivated in a CO.sub.2 incubator for one more
day. Thereafter, DMEM/F12 containing partial peptide of lacritin is
added and, after stimulation for 10 min in a CO.sub.2 incubator,
the medium is recovered. The recovered medium is desalted and
concentrated by ReadyPrep 2D Cleanup Kit (Bio-Rad Laboratories,
Inc., catalog No. 163-2130), dissolved in NuPAGE LDS sample buffer
(Invitrogen, catalog No. NP0007), and denatured by heating at
70.degree. C. for 10 min. An equivalent amount of a sample is
separated in 4-12% NuPAGE Bis-Tris gel, MES buffer at 200 V for 35
min at room temperature, and blotted on PVDF membrane using
Transblot Mini Cell (Bio-Rad Laboratories, Inc.) at 100 V for 60
min. The membrane is subjected to blocking using TTBS containing
0.5% skim milk at room temperature for 30 min and reacted with
5000-fold diluted lactoferrin antibody (Sigma, catalog No. L3262)
overnight at 4.degree. C. After washing with TTBS, anti-rabbit HRP
secondary antibody (Santa Cruz Biotechnology, Inc., catalog No.
sc-2054) is diluted 10000-fold and reacted at room temperature for
60 min. Then, lactoferrin is detected using ECL plus (GE
Healthcare, catalog No. RPN2132).
Experimental Example 6
Anterior Ocular Segment Stimulation Test
[0109] In this Experimental Example, an eye drop containing each
polypeptide was instilled and irritation in the anterior ocular
segment was evaluated.
[0110] Polypeptide 1 (Pyr Lac50-83) ophthalmic solution and
polypeptide 5 (Lac49-83) ophthalmic solution were prepared by
diluting each polypeptide with PBS [obtained by dissolving sodium
chloride 0.9 g (NACALAI TESQUE, INC., catalog No. 31320-05) and
sodium dihydrogen phosphate 0.1 g (NACALAI TESQUE, INC., catalog
No. 31718-15) with distilled water to the total amount of 100 ml
(pH 7.0)] to 1 mM concentration.
[0111] Polypeptide 1 ophthalmic solution, polypeptide 5 ophthalmic
solution or PBS (control group) was instilled 8 times to male
Japanese white rabbit (KITAYAMA LABES Co., Ltd., body weight about
2.0 kg; N=1 in polypeptide 1 administration group and N=2 in
polypeptide 5 administration group) by 100 .mu.L one time, 1 hr
interval. The observations of anterior ocular segment and the
observation of corneal staining plaque with fluorescein (Wako Pure
Chemical Industries, Ltd., catalog No. 213-00092) were performed
before ocular instillation and 30 min after final ocular
instillation. Irritant property in the anterior ocular segment was
evaluated by McDonald-Shadduck method (Hackett, R. B. and McDonald,
T. O., Chapter 44: Assessing Ocular Irritation, Dermatotoxicology
fifth edition, Francis N. Marzulli and Howard I. Maibach, Taylor
& Francis, U.S.A.: 557-567, 1996).
[0112] The anterior ocular segment after ocular instillation was
observed for cornea, iris, and conjunctiva. As a result, as to the
cornea, transparency was normal; cloudiness and vascularization in
the corneal stroma were not found; and corneal staining plaque due
to fluorescein was not observed. As to the iris, optical response
was normal; and iris congestion was not found. As to the
conjunctiva, conjunctival congestion and swelling were not found,
and the conjunctiva was normal. In addition, abnormal secreted
material was not observed. These results correspond to score 0
according to the evaluation by McDonald-Shadduck method (see
below), and abnormality was not observed with both polypeptide 1
ophthalmic solution and polypeptide 5 ophthalmic solution.
Moreover, both ophthalmic solutions showed no abnormality in the
observation of corneal staining plaque. Therefrom it has been
clarified that polypeptide 1 ophthalmic solution and polypeptide 5
ophthalmic solution have high safety.
Criteria For Determining Score 0
[0113] A) Conjunctival congestion; normal, without congestion.
Blood vessels are sometimes observed in the perilimbal region and
the palpebral and bulbar conjunctiva around 12:00 and 6:00 o'clock
positions. B) Conjunctival swelling; normal, without swelling. C)
Secreted material; normal, without secretion. D) Optical response
of iris; normal pupil response. E) Iris congestion; normal. A
congested area of about 1 mm to 2 mm in diameter is sometimes
observed near the pupillary border around the 12:00 to 1:00 o'clock
position and 6:00 to 7:00 o'clock position. F) Transparency of
cornea; normal. G) Level of corneal cloudiness; normal cornea
without cloudiness. H) Vascularization in corneal stroma; no
vascularization. J) Staining of cornea by fluorescein staining; no
fluorescein staining.
Formulation Example 1
Culture Medium for Preparation of Corneal Epithelial Sheet
[0114] Four milliliters of HCGS growth additive (contents: mEGF,
hydrocortisone, insulin, transferrin and BPE, KURABO INDUSTRIES
LTD. catalogue No. KC-6150) and 15 mg of polypeptide 1 are added to
EpiLife medium (corneal epithelial cell basal medium, Cascade
Biologics, catalogue No. M-EPI-500-CA) to give a culture medium
(total amount 500 mL).
Formulation Example 2
Eye Drop Containing Lacritin Partial Peptide
[0115] The eye drop shown below is prepared according to a
conventional method.
TABLE-US-00002 polypeptide 1 0.5 g sodium dihydrogen phosphate 0.1
g sodium chloride 0.9 g sodium hydroxide e.q. sterilized purified
water e.q. total amount 100 mL (pH 7)
INDUSTRIAL APPLICABILITY
[0116] According to the present invention, a novel polypeptide that
promotes adhesion between a cell and extracellular matrix,
particularly adhesion between a corneal epithelial cell and
extracellular matrix, and is stable in aqueous solution can be
provided. Since the polypeptide of the present invention can
promote adhesion between a corneal epithelial cell and
extracellular matrix, and is stable in aqueous solution, a
medicament superior in preservation stability, particularly such
medicament in the form of an aqueous liquid, can be provided.
DESCRIPTION OF SEQUENCE LISTING
[0117] SEQ ID NO: 1 amino acid sequence of pyro-modified partial
peptide of lacritin (Pyr Lac50-83) SEQ ID NO: 2 amino acid sequence
of partial peptide of lacritin (Lac50-83) SEQ ID NO: 3 amino acid
sequence of pyro-modified partial peptide of lacritin (Pyr
Lac50-94) SEQ ID NO: 4 amino acid sequence of peptide of lacritin
(Lac50-94) SEQ ID NO: 5 amino acid sequence of full-length lacritin
(human) SEQ ID NO: 6 amino acid sequence of acetylated partial
peptide of lacritin SEQ ID NO: 7 amino acid sequence of partial
peptide of lacritin (Lac24-56) SEQ ID NO: 8 amino acid sequence of
partial peptide of lacritin (Lac50-65) SEQ ID NO: 9 amino acid
sequence of partial peptide of lacritin (Lac49-83)
SEQUENCE LISTING FREE TEXT
[0118] SEQ ID NO: 1: Xaa shows pyroglutamic acid.
[0119] SEQ ID NO: 3: Xaa shows pyroglutamic acid.
[0120] This application is based on a patent application No.
2009-215030 filed in Japan, the contents of which are incorporated
in full herein.
Sequence CWU 1
1
9134PRTHomo sapiensMOD_RES(1)..(1)Xaa is a pyroglutamate. 1Xaa Gly
Thr Ala Lys Val Thr Ser Ser Arg Gln Glu Leu Asn Pro Leu1 5 10 15Lys
Ser Ile Val Glu Lys Ser Ile Leu Leu Thr Glu Gln Ala Leu Ala 20 25
30Lys Ala234PRTHomo sapiens 2Gln Gly Thr Ala Lys Val Thr Ser Ser
Arg Gln Glu Leu Asn Pro Leu1 5 10 15Lys Ser Ile Val Glu Lys Ser Ile
Leu Leu Thr Glu Gln Ala Leu Ala 20 25 30Lys Ala345PRTHomo
sapiensMOD_RES(1)..(1)Xaa is a pyroglutamate. 3Xaa Gly Thr Ala Lys
Val Thr Ser Ser Arg Gln Glu Leu Asn Pro Leu1 5 10 15Lys Ser Ile Val
Glu Lys Ser Ile Leu Leu Thr Glu Gln Ala Leu Ala 20 25 30Lys Ala Gly
Lys Gly Met His Gly Gly Val Pro Gly Gly 35 40 45445PRTHomo sapiens
4Gln Gly Thr Ala Lys Val Thr Ser Ser Arg Gln Glu Leu Asn Pro Leu1 5
10 15Lys Ser Ile Val Glu Lys Ser Ile Leu Leu Thr Glu Gln Ala Leu
Ala 20 25 30Lys Ala Gly Lys Gly Met His Gly Gly Val Pro Gly Gly 35
40 455138PRTHomo sapiens 5Met Lys Phe Thr Thr Leu Leu Phe Leu Ala
Ala Val Ala Gly Ala Leu1 5 10 15Val Tyr Ala Glu Asp Ala Ser Ser Asp
Ser Thr Gly Ala Asp Pro Ala 20 25 30Gln Glu Ala Gly Thr Ser Lys Pro
Asn Glu Glu Ile Ser Gly Pro Ala 35 40 45Glu Pro Ala Ser Pro Pro Glu
Thr Thr Thr Thr Ala Gln Glu Thr Ser 50 55 60Ala Ala Ala Val Gln Gly
Thr Ala Lys Val Thr Ser Ser Arg Gln Glu65 70 75 80Leu Asn Pro Leu
Lys Ser Ile Val Glu Lys Ser Ile Leu Leu Thr Glu 85 90 95Gln Ala Leu
Ala Lys Ala Gly Lys Gly Met His Gly Gly Val Pro Gly 100 105 110Gly
Lys Gln Phe Ile Glu Asn Gly Ser Glu Phe Ala Gln Lys Leu Leu 115 120
125Lys Lys Phe Ser Leu Leu Lys Pro Trp Ala 130 135634PRTHomo
sapiensMOD_RES(1)..(1)ACETYLATION 6Gln Gly Thr Ala Lys Val Thr Ser
Ser Arg Gln Glu Leu Asn Pro Leu1 5 10 15Lys Ser Ile Val Glu Lys Ser
Ile Leu Leu Thr Glu Gln Ala Leu Ala 20 25 30Lys Ala733PRTHomo
sapiens 7Glu Ile Ser Gly Pro Ala Glu Pro Ala Ser Pro Pro Glu Thr
Thr Thr1 5 10 15Thr Ala Gln Glu Thr Ser Ala Ala Ala Val Gln Gly Thr
Ala Lys Val 20 25 30Thr816PRTHomo sapiens 8Gln Gly Thr Ala Lys Val
Thr Ser Ser Arg Gln Glu Leu Asn Pro Leu1 5 10 15935PRTHomo sapiens
9Val Gln Gly Thr Ala Lys Val Thr Ser Ser Arg Gln Glu Leu Asn Pro1 5
10 15Leu Lys Ser Ile Val Glu Lys Ser Ile Leu Leu Thr Glu Gln Ala
Leu 20 25 30Ala Lys Ala 35
* * * * *